Equine rhinopheumonitis and equine abortion are commonly recognised diseases of horses caused by two distinct but antigenically related viruses that are designated equine herpesvirus 4 and equine herpesvirus 1, known as EHV4 and EHV1 respectively. Because the viruses are related antigenically it has not been possible to date by serological examination (blood test), to determine whether a horse has been infected with either or both EHV4 or EHV1. For example, if a horse had been infected with EHV4 as a foal it would develop antibodies in its serum that would react with not only EHV4 but with EHV1 as well, so one would not know that such a foal had been infected with only EHV4.
However, since 1981 it has been repeatedly Shown that the restriction endonuclease fingerprints of the two viruses are distinctly different with respiratory isolates and fetal isolates almost invariably typing as EHV4 and EHV1 respectively. The availability of specific monoclonal antibodies (MAbs) directed to either EHV4 or EHV1 has also allowed consistent and specific typing of isolates of the two viruses.
The major significance in developing a specific antibody test relates to the fact that both these herpesviruses are believed, after primary infection, to establish a persistent, latent and life-long infection. Either virus may from time to time be reactivated from the latent state (just as is the case with recurrent cold sores in humans infected with herpes simplex virus); the virus, reactivated from the latent state, will usually cause recurrent disease in the host horse but more importantly such a horse will either directly or indirectly by contact act as a source of infection for other horses. In this way, for EHV4, there is usually in the annual foal crop born on a farm an annual round of respiratory disease ("snotty" noses). Such an occurrence is almost am accepted part of breeding horses. Occasionally foals become severely affected and require treatment or die because of severe secondary complications such as bacterial pneumonia.
While the natural history of EHV1 is less clearly understood, there is an assumption that the virus does establish persistent, lifelong latent, infections. Upon reactivation there may be a further bout of respiratory disease. However, a far more serious consequence for other horses infected by contact with the first horse (index case) occurs on breeding farms when a pregnant mare in a paddock reactivates the virus and transmits it to other in-contact pregnant mares. The index case mare may herself abort or cause abortion in one or more in contact mares. An aborted foetus and the foetal membranes and fluids are heavily infected with EHV1 and contaminate the site where abortion occurs. Other mares in the paddock, being naturally curious, come to the site of abortion and sniff the foetus and membranes. In this way, often close to 100% of the mares in the paddock become infected and abort within 10 or 20 days causing what is commonly known as an "abortion storm". Such outbreaks of EHV1 abortion are of considerable economic importance to the equine, particularly thoroughbred and standardbred, industries worldwide.
There is a need for accurate, type-specific serological surveillance of horses for the presence of EHV4 and/or EHV1 antibodies to assist in our understanding of the epidemiology of these viruses, particularly EHV1. Presently, however, EHV1 or EHV4 antibodies in polyclonal serum cannot be differentiated because of the extensive antigenic cross-reactivity between the two viruses. The availability of such a specific serological test would also have profound implications in the control, perhaps eradication, of EHV1 and in the selection of candidate horses for vaccination.
The antibody responses of the horse to these viruses is largely directed to the envelope glycoproteins. EHV1 homologues to nine of the ten recognized herpes simplex virus 1 (HSV1) glycoproteins have been identified from DNA sequence analyses; gB, gC, gD, gE, gG, gH, gI, gK, and gL. The remaining HSV1 glycoprotein, gJ, has a positional counterpart in the US region of EHV1, gene 71, although these two genes do not show any significant homology. Also, EHV1 possesses at least three other glycoproteins designated gp2, gp10, which are the homologues of the HSV1 tegument proteins VP13/14 and gp21/22a. In the case of EHV4, which unlike EHV1 has only been partially sequenced, glycoprotein genes encoding gB, gC, gG and gH homologues have been identified which show amino acid identities of 89%, 79%, 58% and 85% respectively with their EHV1 counterparts. Homologous EHV4 and EHV1 genes map to collinear positions in their respective genomes.
EHV1 glycoproteins gp2, gp10, gC (gp13), gB (gp14), gD (gp18) and gp21/22a have been definitively identified by SDS-PAGE as have EHV4 glycoproteins gp2, gp10, gC (gp13), gB (gp14), gD (gp18) and gG. Using combinations of EHV1/EHV4 MAbs and polyclonal post-EHV1 only or post-EHV4 only horse sera it has been shown that each of the glycoproteins possesses both type-common and type-specific epitopes The two exceptions are gp21/22a which has been relatively poorly studied and EHV4 gG which appears to elicit a type-specific serological response.
To date, glycoprotein G (gG) homologues of other herpesviruses have been used as a basis for diagnostic testing and clinical application. In particular, gG (alternatively called gX) of pseudorabies virus (PRV) has been used in the development of gG deletion mutant vaccines linked to a diagnostic test for PRV of pigs.
Also gG of human herpes simplex viruses (HSV) 1 and 2 are different enough to allow tests such as ELISA to be developed for the detection of antibodies to either HSV1 or 2 in the serum of humans.
However, in spite of these data for PRV and HSV 1 and 2 it could not have been predicted that the same glycoprotein, namely glycoprotein G of EHV4 and EHV1, would serve a role in diagnosis and vaccine development. For PRV, only a single virus is involved, so the question of distinguishing between two viruses is not relevant. For HSV 1 and 2, while two viruses are involved, the gG proteins are different in very different ways from EHV4 and EHV1 gGs. In particular, the molecular sizes of unglycosylated HSV1 and HSV2 gGs differ greatly with HSV1 at 26 K and, HSV2 at 77 K, whereas the sizes of unglycosylated EHV4 and EHV1 gG differ only slightly at 48 K and 45 K respectively. It is reasoned that the type-specificity of the entire HSV1 and 2 gG proteins resulted from a major deletion in the case of the HSV1 gG gene whereby some 1383 nucleotides have been "lost" from a total gene (in the case of HSV2) of 2097 nucleotides. The loss of more than half the coding sequence of HSV1 gG results in a non glycosylated protein of only 26 K vs 77 K for HSV2 gG. While it is recognised that a positive ELISA is required for the differentiation of humans infected with either HSV1 or 2 it could have been reasoned that if the two genes were of approximately the same size then the two gGs would be cross-reactive. For EHV4 and EHV1 it is shown that while the two gGs are approximately the same size they are still type-specific. This could not have been predicted from prior art (HSV) material or from sequence data alone. On the later point the fact that EHV4 and EHV1 gG are 58% similar at the amino acid level it could in fact have been predicted that cross-reactive epitopes would almost certainly have existed and hence gG could not have been used in the concurrently described invention to differentiate the two equine viruses. The findings of the current invention are based on the analysis of a specifically selected set of horse serums for which the previous infection/vaccination history of particular horses was known, Detailed analysis of these serums show that cross-reactive epitopes are either not present or are not important in eliciting an antibody response in the natural host. Such a highly distinctive properly of these epitopes was quite unexpected and has allowed the development of the instant invention.